Methods of inhibiting metastasis

ABSTRACT

Methods of inhibiting metastasis or maintenance of various cancers are provided. In particular, the method makes use of fact that certain trafficking of cancers depend upon identified proteins, which serve as markers. Additional methods of screening are also provided.

The present application is a continuation of U.S. patent applicationSer. No. 11/197,711, filed Aug. 4, 2005; which is a divisionalapplication of U.S. patent application Ser. No. 09/721,613, filed Nov.22, 2000, which is now U.S. Pat. No. 6,949,243; which claims the benefitof U.S. provisional patent application No. 60/225,562, filed Aug. 14,2000; and U.S. provisional patent application No. 60/167,519, filed Nov.24, 1999; each of which is incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates generally to methods of inhibiting metastasis ofvarious cancers. It also provides methods for screening for variousproteins which will exhibit similar biological activity.

BACKGROUND

Even if a primary cancer is completely eliminated, a malignant tumorwill often be metastatic. The formation of metastases of malignanttumors, initiated from a primary tumor at more or less remote locationsof the body, is one of the most serious effects of cancer and one forwhich a satisfactory treatment protocol is currently unavailable. Cancertumor metastasis is responsible for most therapeutic failures when thedisease is treated, as patients succumb to the multiple tumor growth.Important tumors include, e.g., carcinomas, including breast, head andneck, lung, colon, prostate, and melanomas. See, e.g., Bertino, et al.(eds. 1996) Encyclopedia of Cancer Academic Press; Devita, et al. (eds.1997) Cancer: Principles & Practice of Oncology Lippincott, Williams andWilkins; Devita (1997) Principles and Practice of Oncology LippincottWilliams and Wilkins; Cavalli, et al. (1996) Textbook of MedicalOncology Dunitz Martin Ltd; Horwich (ed. 1995) Oncology: AMultidisciplinary Textbook Lippincott-Raven; Peckham, et al. (eds. 1995)Oxford Textbook of Oncology Oxford Univ. Press; Mendelsohn, et al.(1995) The Molecular Basis of Cancer Saunders, Philadelphia; and McArdle(1990) Surgical Oncology: Current Concepts and PracticeButterworth-Heinemann.

The extent to which metastasis occurs varies with the individual type oftumor. Melanoma, breast cancer, lung cancer, colon cancer, and prostatecancer are among the types of cancer that are prone to metastasize. Whenmetastasis takes place, the metastases can form at a variety of sites inthe body, with lymph nodes, lungs, liver, brain and bone marrow beingthe more common sites.

The currently available methods of cancer therapy such as surgicaltherapy, radiotherapy, chemotherapy, and immunobiological methods haveeither been of limited success in preventing metastasis or these methodsgive rise to serious and undesirable side effects.

In many clinically diagnosed solid tumors (in which the tumor is alocalized growth) surgical removal is considered the prime means oftreatment. However, many times after surgery and after some delayperiod, the original tumor is observed to have metastasized so thatsecondary sites of cancer invasion have spread throughout the body andthe patient subsequently dies of the secondary cancer growth. Reportsindicate that in individuals with resectable tumors, primary tumorgrowth or local recurrence is not often the cause of death. Instead, atpresent, nearly 40% of cancer victims with operable tumors ultimatelysuccumb to metastatic disease following surgery.

Although chemotherapy is widely used in the treatment of cancer, it is asystemic treatment based usually on the prevention of cellproliferation. Accordingly, chemotherapy is a non-specific treatmentmodality affecting all proliferating cells, including normal cells,leading to undesirable and often serious side effects, e.g.,immunosuppression, pancytopenia (growth inhibition of bone marrow cellswith anemia, thrombocytopenia, and leukopenia), diarrhea, nausea andalopecia (hair loss).

Often, the existing systemic treatments have proven to have littleeffect on macrometastases already residing in remote organs (lung,liver, bone marrow, or brain). Patients are often killed with metastaticcancers provoked by metastasis of cancer cells. A method for effectivelysuppressing the metastasis of the cancer cells has not been established,and a medicine having a cancer cell metastasis suppressing effect hasnot yet been made commercially available.

Thus, the need exists for methods of inhibiting tumor metastasis. Inparticular, methods which inhibit metastasis without causing seriousside effects are much desired.

SUMMARY OF THE INVENTION

The present invention is based, upon the hypothesis that signalingthrough chemokine receptors may be an important attractant mechanism formetastasizing cancer cells. Data supporting such model have beencollected. Alternatively, the specific expression by tumor cells ofchemokine receptors may allow for prevention of progression of growth,or even shrinkage, e.g., by chemokine receptor targeted therapeuticagents, alone or in combination with other anticancer therapies.

The present invention provides methods of inhibiting metastasis of acell, e.g., a tumor cell, the method comprising blocking signaling of achemokine receptor on the cell. Preferably, the metastasis is organspecific, e.g., to the lymph node, bone marrow, or skin, or the cell isa carcinoma cell, including a breast, head and neck, melanoma, orprostate. In other embodiments, the chemokine receptor is CCR7, CXCR4,or CCR10. The blocking of signaling can be with an antibody against thechemokine receptor, a mutein antagonist of the ligand, or a drug whichinhibits signaling of the chemokine receptor, e.g., pertussis toxin.Thus, e.g., when the chemokine receptor is CCR7, the blocking may bewith: an antibody against FSEC, CKβ9, or CKβ11; or an antagonist muteinof FSEC, CKβ9, or CKβ11; when the chemokine receptor is CXCR4, theblocking may be with: an antibody against SDF-1; or an antagonist muteinof SDF-1; and when the chemokine receptor is GPR2, the blocking may bewith: an antibody against CTACK or Vic; or an antagonist mutein of CTACKor Vic. Use of toxic conjugates to target drugs to chemokine receptorexpressing tumors may also be effected.

Typically, the method is combined with another treatment for cancer,e.g., chemotherapy, radiation therapy, immunotherapy, or surgery. Oftenthe method is applied after such treatment, but it may be prophylactic.And the treatment may be directly to affect primary tumor progression orgrowth.

Alternatively, the invention provides methods of screening for chemokinereceptors on metastatic or primary tumor cells, comprising identifyingwhich chemokine receptors are expressed on the cell. The identifying maybe by, e.g., antibody labeling, ligand testing, or PCR analysis; or maybe useful in determination of therapeutic strategy. The labeling mayallow for directed cell killing, e.g., with toxic conjugates, or withabsorption reagents allowing for absorption of energy or binding oftoxic compounds.

Yet another embodiment includes a composition comprising an anti-tumoragent and a chemokine receptor antagonist or label. In such embodiments,the antagonist or label may be an antibody against FSEC, CKβ9, or CKβ11;an antagonist mutein of FSEC, CKP9, or CKβ11; an antibody against SDF-1;an antagonist mutein of SDF-1; an antibody against CTACK or Vic; anantagonist mutein of CTACK or Vic; an antibody against the chemokinereceptor; or a drug which inhibits signaling of the chemokine receptorincluding pertussis toxin. Methods using such compositions are provided,e.g., methods of treating a cancer in an animal, metastasizing orprimary, comprising administering to the animal an effective amount ofthe composition.

DETAILED DESCRIPTION OF THE INVENTION Outline I. General II. ChemokineAgonists and Antagonists

A. Ligands and Variants

B. Antibodies

C. Other Molecules

III. Immunoassays IV. Uses I. General

The invention is based, in part, on the surprising discovery of acorrelation suggesting that chemokines may be important mediators ofcancer metastasis. In particular, metastatic tumors have been analyzedfor chemokine receptor expression. Various primary tumors have also beenevaluated for chemokine receptor expression, and the identification ofthe receptors as markers may have therapeutic value.

Organ specific metastatic tumors appear to express specific chemokinereceptors. These chemokine receptors appear to correspond to the ligandexpression of the common metastatic target organs of the various cancersevaluated. Moreover, in evaluation of the primary tumors, the chemokinereceptor expression patterns also match the ligands expressed by themetastatic target organs. Thus, those metastatic tumor cells likely weredirected to the target organ, in part, by the chemokines expressed bythose target organs. If so, then metastasis will be responsive toblocking of chemokine mediated trafficking and organ targeting.

In addition, identification of various chemokine receptors as markersfor specific tumor types provides the possibility of using them asmarkers to target appropriate therapeutics. Toxic conjugates, specificlocalization of radiation or energy absorbents, or means to attractanticancer drugs to the sites of primary tumors will be useful.

The chemokines are a sub-family of chemoattractant cytokines that wereclassically characterized by their ability to mediate leukocytetrafficking or migration by binding to specific G-protein linked seventransmembrane spanning receptors, or GPCRs. Chemokines are divided intofour groups based on the primary sequence of the first two cysteines:the CXC, CC, C, and CX3C families.

The chemokine receptors are typically members of the superfamily ofG-protein coupled (or linked) receptors (GPCR, or GPLR). As a class,these receptors are integral membrane proteins characterized by aminoacid sequences which contain seven hydrophobic domains. See, e.g.,Ruffolo and Hollinger (eds. 1995) G-Protein Coupled TransmembraneSignaling Mechanisms CRC Press, Boca Raton, Fla.; Watson and Arkinstall(1994) The G-Protein Linked Receptor FactsBook Academic Press, SanDiego, Calif.; Peroutka (ed. 1994) G Protein-Coupled Receptors CRCPress, Boca Raton, Fla.; Houslay and Milligan (1990) G-Proteins asMediators of Cellular Signaling Processes Wiley and Sons, New York,N.Y.; and Dohlman, et al. (1991) Ann. Rev. Biochem. 60:653-688. Thesehydrophobic domains are predicted to represent transmembrane spanningregions of the proteins. These GPCRs are found in a wide range oforganisms and are typically involved in the transmission of signals tothe interior of the cell, e.g., through interaction, e.g., withheterotrimeric G-proteins. They respond to a wide and diverse range ofagents including lipid analogs, amino acid derivatives, small peptides,and other molecules. The chemokine ligands for the receptors typicallyinitiate a calcium flux upon binding to the receptor, and the calciumflux is typically pertussis toxin sensitive. Besides chemoattractantproperties, chemokines have been shown to induce other biologicalresponses, e.g., modulation of second messenger levels such as Ca⁺⁺;inositol phosphate pool changes (see, e.g., Berridge (1993) Nature361:315-325 or Billah and Anthes (1990) Biochem. J. 269:281-291);cellular morphology modification responses; phosphoinositide lipidturnover; possible antiviral responses; and others.

The best known biological functions of chemokine molecules relate tochemoattraction of leukocytes. It would not be peculiar that traffickingof other cell types, e.g., primary tumor cells expressing thosechemokine receptors, would likewise be directed by chemokines tospecific organs. See, e.g., Youngs, et al. (1997) Int. J. Cancer71:257-266; and Kleeff, et al. (1999) Int. J. Cancer 81:650-657.

A significant panel of tumor cell lines of various types were collected,most available from the ATCC. These included breast carcinoma, head andneck, melanoma, and prostate cancer cell lines. The cell lines wereanalyzed for expression of a number of chemokine receptors byquantitative PCR techniques. By this analysis, chemokine receptorexpression was generally very low, with the exception of the CCR7 (seeGenBank L31581), the CXCR4 (see GenBank X71635), and the GPR2 (seeGenBank U13667; also designated CCR10) receptors. The breast cancerlines generally expressed the CCR7, CXCR4, and GPR2 receptors. The headand neck tumor lines generally expressed the CCR7 receptor. The melanomacell lines generally expressed the CCR7, CXCR4, and GPR2 receptors. Insummary, the CCR7 receptor was expressed by the breast cancer, head andneck, and melanoma cell lines; the CXCR4 receptor was expressed by thebreast cancer and melanoma cell lines; and the GPR2 receptor wasexpressed by breast cancer and melanoma cell lines. The prostatecarcinoma lines expressed CCR7, CXCR4, CCR8, and STRL33; the breastcancer cell lines also expressed CCR8 and STRL33.

Each of these receptors has been matched with chemokine ligands. Thechemokines MIP-3β (GenBank U77180; see Coleman, et al. WO9622374A1(FSEC)) and CKβ9 (GenBank W17274; see Adams and Li WO9606169A1) are theligands for the CCR7 receptor. Both of these chemokines are expressed bylymph nodes, which is probably why all three tumor cell types oftenmetastasize to the lymph node. The chemokine SDF-1 (stromal cell derivedfactor 1; GenBank L12030 and AA620142; see Lacey, et al. (1997) Proc.Nat'l Acad. Sci. USA 94:9842-9847, and Aiuti, et al. (1997) J. Exp. Med.185:111-120) is the ligand for the CXCR4 receptor. This chemokine isexpressed by bone marrow stromal cells, which is probably why breastcancers often metastasize to the bone marrow. The CXCR4 has also beenimplicated in liver, lung, and lymph node metastasis because SDF-1 isalso expressed by stromal cells of these organs. The chemokines CTACK(CCL27; GenBank U13667; see Hedrick, et al. WO9823750A2) and Vic (CCL28;GenBank R38459; see Hedrick, et al. WO9823750A2) are the ligands for theGPR2 receptor. These chemokines are expressed in the skin, whichprobably explains why melanomas metastasize to the skin.

Besides metastatic effects, the SDF-1 and CTACK may have importantcontributions to primary tumor formation or progression of growth and/orangiogenesis. This occurs with certain other tumors, and there is reasonto believe this may be true in melanomas.

In a similar fashion for other types of cancers, chemokine receptorswhich are expressed by primary tumors seem generally to have ligandswhich are expressed in the target organs for frequent metastasis. Thismethod may be used to confirm the hypothesis in other tumor cell types.Looking at primary tumors, analysis of chemokine receptors will indicatewhat chemokines those cells are likely to be chemoattracted by. Thus,blockage of metastasis of those primary tumors should be mediated byblockage of such chemoattraction. The blockage may be effected by ligandantagonists or receptor antagonists. Such may be ligand muteinantagonists, antibody antagonists to ligand or receptor, or drugs, e.g.,small molecules, which block chemoattraction.

The most common other primary tumor types include, e.g., prostatecancer, gastrointestinal (including colon) cancer, and lung cancer.Prostate cancers tend to metastasize to lymph nodes and bone marrow,suggesting that the CCR7 and CXCR4 receptors are involved, mediated bytheir respective ligands. Gastrointestinal cancers tend to metastasizeto lymph nodes and liver, suggesting that the CCR7 and CCR6 receptorsare involved. The ligand for the CCR6 receptor is the chemokine MIP-3α.Lung cancers tend to metastasize to the lymph nodes, bone marrow, andbrain, suggesting that the CCR7, CXCR4, and V28 chemokine receptors areinvolved, mediated by their respective chemokine ligands. The ligand forthe V28 receptor is the chemokine CX3C (neurotactin). The presentinvention teaches what antagonists will have effects on certain types ofprimary tumor metastasis or progression.

Conversely, chemokines expressed by target tissues for metastasis arelikely mediators of chemoattraction. Thus, metastasis may be blocked byinhibiting the chemoattraction or tumor progression may be targeted,e.g., at the chemokine receptors as markers. It is likely thatmetastasis to the lymph nodes is mediated by the CCR7 and CXCR4receptors, to the bone marrow by the CXCR4 receptor, to the skin by theGPR2 receptor, to the liver by the CXCR4 CCR6 receptors, to the brain bythe V28 receptor, and to the lung by the CXCR4 receptor. Likewise, thepresent invention teaches what antagonists will have effects onmetastasis to specific organs or markers for primary tumors. And thespecific expression of chemokine receptors on primary tumors allow forspecific targeting of therapeutic agents to those sites.

In either case, mouse models will be useful in confirmation of theteachings. The testing of other primary tumor types or target organs forreceptor/chemokine pairings responsible for the metastasis or primarytumor progression, e.g., growth and/or angiogenesis, will be continued,e.g., other types of carcinomas, sarcomas, etc. Primary tumorprogression may be also targeted.

II. Chemokine Agonists and Antagonists

Chemokine ligands for the receptors have been described. Variousagonists and antagonists of the natural ligands or receptors can beproduced. Receptor binding assays can be developed. See, e.g., Bieri, etal. (1999) Nature Biotechnology 17:1105-1108, and accompanying note onpage 1060. Calcium flux assays may be developed to screen for compoundspossessing antagonist activity. Migration assays may take advantage ofthe movement of cells through pores in membranes, which can form thebasis of antagonist assays. Chemotaxis may be measured thereby.Alternatively, chemokinetic assays may be developed, which measure theinduction of kinetic movement, not necessarily relative to a gradient,per se.

A. Chemokine Ligands and Variants

Chemokine agonists will exhibit some or all of the signaling functionsof the chemokine, e.g., binding, inducing a Ca++ flux, andchemoattracting appropriate receptor bearing cells. Conversely,antagonists will block the signaling and/or effector biology. Variousmammalian chemokine sequences may be evaluated to determine whatresidues are conserved across species, suggesting what residues may bechanged without dramatic effects on biological activity. Alternatively,conservative substitutions in certain regions of the molecule aresomewhat more likely to maintain receptor binding activity, while otherregions will more likely affect signal transduction. Standard methodsfor screening mutant or variant chemokine polypeptides will determinewhat sequences will be useful therapeutic antagonists.

In addition, certain nucleic acid expression methods may be applied. Forexample, in certain contexts, it may be useful to transfect cells withvarious nucleic acids which will be expressed, as appropriate. Variouspromoters may be operably linked to the gene, thereby allowing forregulated expression, e.g., suppression.

Antagonist activity may be tested or screened for using well knownmethods. Tests for ability to antagonize chemokine binding, calciumflux, or chemoattractant activity can be developed. Various ligandhomologs can be created which retain receptor binding capacity, but lacksignaling capability, thus serving as competitive binding molecules.Small molecules may also be screened for ability to antagonize chemokinefunction, e.g., chemoattraction, receptor binding, Ca++ flux, and othereffects mediated by chemokine. See generally Gilman, et al. (eds. 1990)Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8thEd., Pergamon Press; Remington's Pharmaceutical Sciences, 17th ed.(1990), Mack Publishing Co., Easton, Pa., each of which is incorporatedherein by reference. Agonists or antibodies may function as means totarget, e.g., for labeling or specific localization, primary tumortypes.

B. Antibodies

The present invention provides for the use of an antibody or bindingcomposition which specifically binds to chemokine or receptor,preferably mammalian, e.g., primate, human, cat, dog, rat, or mouse, andneutralizes the ability of the chemokine to mediate its signal.Non-neutralizing antibodies may be useful for labeling or localization.Antibodies can be raised to various chemokine ligand or chemokinereceptor proteins, including individual, polymorphic, allelic, strain,or species variants, and fragments thereof, either in their naturallyoccurring (full-length) forms or in their recombinant forms.Additionally, antibodies can be raised to chemokine ligands orpolypeptides in either their native (or active) forms or in theirinactive, e.g., denatured, forms, which may neutralize ligand capacityto mediate its signal. Antibodies may block the interaction of theligand with its receptor, e.g., by steric hindrance, or may serve asreagents allowing labeling or localization specifically to where cognateantigen is expressed.

In particular, receptor antagonists may be produced by making antibodieswhich bind to the receptor and block ligand binding. With theidentification of a receptor for the cytokine, antibodies to thereceptor may be selected, e.g., for those which block the binding of, orsignaling induced by, ligand. Or targeting reagents can be produced.

A number of immunogens may be selected to produce antibodiesspecifically reactive, or selective for binding, with ligand or receptorproteins. Recombinant protein is a preferred immunogen for theproduction of monoclonal or polyclonal antibodies. Naturally occurringprotein, from appropriate sources, e.g., primate, rodent, etc., may alsobe used either in pure or impure form. Synthetic peptides, made usingthe ligand or receptor protein sequences, may also used as an immunogenfor the production of antibodies. Recombinant protein can be expressedand purified in eukaryotic or prokaryotic cells as described, e.g., inColigan, et al. (eds. 1995 and periodic supplements) Current Protocolsin Protein Science John Wiley & Sons, New York, N.Y.; and Ausubel, et al(eds. 1987 and periodic supplements) Current Protocols in MolecularBiology, Greene/Wiley, New York, N.Y. Naturally folded or denaturedmaterial, perhaps expressed on cell surfaces, can be used, asappropriate, for producing antibodies. Either monoclonal or polyclonalantibodies may be generated, e.g., for subsequent use in immunoassays tomeasure the protein, for immunopurification methods, or for targetingmethods.

Methods of producing polyclonal antibodies are well known to those ofskill in the art. Typically, an immunogen, preferably a purifiedprotein, is mixed with an adjuvant and animals are immunized with themixture. The animal's immune response to the immunogen preparation ismonitored by taking test bleeds and determining the titer of reactivityto, e.g., the ligand or receptor, protein or polypeptide of interest.For example, when appropriately high titers of antibody to the immunogenare obtained, usually after repeated immunizations, blood is collectedfrom the animal and antisera are prepared. Further fractionation of theantisera to enrich for antibodies reactive to the protein can beperformed, if desired. See, e.g., Harlow and Lane Antibodies, ALaboratory Manual; or Coligan (ed.) Current Protocols in Immunology.Immunization can also be performed through other methods, e.g., DNAvector immunization. See, e.g., Wang, et al. (1997) Virology228:278-284. Affinity purification, or absorptions, can be used toselect for desired specificity of binding.

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Typically, spleen cells from an animalimmunized with a desired antigen are immortalized, commonly by fusionwith a myeloma cell. See, Kohler and Milstein (1976) Eur. J. Immunol.6:511-519. Alternative methods of immortalization include transformationwith Epstein Barr Virus, oncogenes, or retroviruses, or other methodsknown in the art. See, e.g., Doyle, et al. (eds. 1994 and periodicsupplements) Cell and Tissue Culture: Laboratory Procedures, John Wileyand Sons, New York, N.Y. Colonies arising from single immortalized cellsare screened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according, e.g., to the general protocol outlined by Huse,et al. (1989) Science 246:1275-1281.

Antibodies or binding compositions, including binding fragments andsingle chain versions, against predetermined fragments of receptor orligand polypeptides can be raised by immunization of animals withconjugates of the fragments with carrier proteins as described above.Monoclonal antibodies are prepared from cells secreting the desiredantibody. These antibodies can be screened for binding to normal ordefective ligand protein, or screened for capacity to block cell ligandmediated flux, chemoattraction, or chemokinetic activity. Thesemonoclonal antibodies will usually bind with at least a K_(D) of about 1mM, more usually at least about 300 μM, typically at least about 10 μM,more typically at least about 30 μM, preferably at least about 10 μM,and more preferably at least about 3 μM or better.

In some instances, it is desirable to prepare monoclonal antibodies(mAbs) from various mammalian hosts, such as mice, rodents, primates,humans, etc. Description of techniques for preparing such monoclonalantibodies may be found in, e.g., Stites, et al. (eds.) Basic andClinical Immunology (4th ed.) Lange Medical Publications, Los Altos,Calif., and references cited therein; Harlow and Lane (1988) Antibodies:A Laboratory Manual CSH Press; Goding (1986) Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press, New York, N.Y.; andparticularly in Kohler and Milstein (1975) Nature 256:495-497, whichdiscusses one method of generating monoclonal antibodies. Summarizedbriefly, this method involves injecting an animal with an immunogen. Theanimal is then sacrificed and cells taken from its spleen, which arethen fused with myeloma cells. The result is a hybrid cell or“hybridoma” that is capable of reproducing in vitro. The population ofhybridomas is then screened to isolate individual clones, each of whichsecrete a single antibody species to the immunogen. In this manner, theindividual antibody species obtained are the products of immortalizedand cloned single B cells from the immune animal generated in responseto a specific site recognized on the immunogenic substance.

Other suitable techniques involve selection of libraries of antibodiesin phage or similar vectors. See, e.g., Huse, et al. (1989) “Generationof a Large Combinatorial Library of the Immunoglobulin Repertoire inPhage Lambda,” Science 246:1275-1281; and Ward, et al. (1989) Nature341:544-546. The polypeptides and antibodies of the present inventionmay be used with or without modification, including chimeric orhumanized antibodies. Frequently, the polypeptides and antibodies willbe labeled by joining, either covalently or non-covalently, a substancewhich provides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentmoieties, chemiluminescent moieties, magnetic particles, and the like.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Also, recombinant immunoglobulins may be produced, see,Cabilly, U.S. Pat. No. 4,816,567; and Queen, et al. (1989) Proc. Nat'lAcad. Sci. USA 86:10029-10033; or made in transgenic mice, see Mendez,et al. (1997) Nature Genetics 15:146-156.

Antibody binding compounds, including binding fragments, of thisinvention can have significant diagnostic or therapeutic value. They canbe useful as non-neutralizing binding compounds and can be coupled totoxins or radionuclides so that when the binding compound binds to theantigen, a cell expressing it, e.g., on its surface, is killed. Further,these binding compounds can be conjugated to drugs or other therapeuticagents, either directly or indirectly by means of a linker, and mayeffect drug targeting.

C. Other Molecules

Antibodies are merely one form of specific binding compositions. Otherbinding compositions, which will often have similar uses, includemolecules that bind with specificity to the receptor, e.g., CCR7, CXCR4,or GPR2, in a binding partner-binding partner fashion, anantibody-antigen interaction, a ligand:receptor interaction with orwithout signaling, or in a natural physiologically relevantprotein-protein interaction, either covalent or non-covalent, e.g.,proteins which specifically associate with chemokine receptor protein.The molecule may be a polymer, or chemical reagent. A functional analogmay be a protein with structural modifications, or may be a structurallyunrelated molecule, e.g., which has a molecular shape which interactswith the appropriate binding determinants. Application of, e.g.,Systematic Evolution of Ligand by Exponential Enrichment (SELEX)technology, methods are available to select specific binding constructsfor desired targets. See, e.g., Colas, et al. (1996) Nature 380:548-550;Cohen, et al. (1998) Proc. Nat'l Acad. Sci. USA 95:14272-14277; Kolonin,et al. (1998) Proc. Nat'l Acad. Sci. USA 95:14266-14271; Famulok, et al.(1998) Curr. Opin. Chem. Biol. 2:320-327; and Eaton, et al. (1997)Bioorg. Med. Chem. 5:1087-1096.

Drug screening can be performed to identify compounds having capacity tobind to receptor, and/or to block chemoattraction to chemokine,Ca++flux, or the natural interaction with ligand. Subsequent biologicalassays can then be utilized to determine if the compound has intrinsicbinding or blocking activity, e.g., an antagonist. Pertussis toxin isone compound known to block certain functions of the chemokine receptorsignaling in a manner distinct from the ligand/receptor interaction.Mutein antagonists may be developed which maintain receptor binding butlack signaling.

Structural studies of the ligands will lead to design of new variants,particularly analogs exhibiting antagonist properties on the receptor.This can be combined with previously described screening methods toisolate muteins exhibiting desired spectra of activities. Or ligands maybe used to target or label receptor bearing cells, e.g., primary tumors.

As receptor specific binding molecules are provided, also included aresmall molecules identified by screening procedures. In particular, it iswell known in the art how to screen for small molecules which interfere,e.g., with ligand binding to the receptor, often by specific binding tothe receptor and blocking of binding by natural ligand. See, e.g.,meetings on High Throughput Screening, International BusinessCommunications, Southborough, Mass. 01772-1749. Such molecules maycompete with natural ligands, and selectively bind to the respectivechemokines or CCR7, CXCR4, or GPR2 receptors. Similarly, assays may bedeveloped which can screen for blockage of downstream signaling pathwaysof the chemokine signaling pathways.

III. Immunoassays

Immunoassays are valuable in diagnosing those cancers which will beresponsive or non-responsive to treatments, as described. Qualitative orquantitative measurement of a particular protein can be performed by avariety of immunoassay methods. For a review of immunological andimmunoassay procedures in general, see Stites and Terr (eds. 1991) Basicand Clinical Immunology (7th ed.). Moreover, the immunoassays of thepresent invention can be performed in many configurations, which arereviewed extensively in, e.g., Maggio (ed. 1980) Enzyme Immunoassay CRCPress, Boca Raton, Fla.; Tijan (1985) “Practice and Theory of EnzymeImmunoassays,” Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers B.V., Amsterdam; Harlow and LaneAntibodies: A Laboratory Manual, supra; Chan (ed. 1987) Immunoassay: APractical Guide Academic Press, Orlando, Fla.; Price and Newman (eds.1991) Principles and Practice of Immunoassays Stockton Press, NY; andNgo (ed. 1988) Non-isotopic Immunoassays Plenum Press, NY.

In particular, the present invention provides various primary ormetastatic cancers susceptible to analysis or diagnosis by evaluatingexpression of select chemokine receptors. For example, the likelihood ofmetastasis would be evaluated by the numbers or types of cellsexpressing these chemokine receptors, making the cell susceptible tochemoattraction by the matching ligand. Prophylactic treatment may beuseful to prevent the recruitment of such tumors to remote metastatictargets, or to label specifically those targets while still small.Alternatively, early targeting of primary tumors may be effected withthe labeling reagents.

Immunoassays for measurement of receptor proteins or peptides can beperformed by a variety of methods known to those skilled in the art. Inbrief, immunoassays to measure the protein can be either competitive ornoncompetitive binding assays. In competitive binding assays, the sampleto be analyzed competes with a labeled analyte for specific bindingsites on a capture agent bound to a solid surface. Preferably thecapture agent is an antibody specifically reactive with receptorproteins produced as described above. The concentration of labeledanalyte bound to the capture agent is inversely proportional to theamount of free analyte present in the sample.

In a competitive binding immunoassay, typically the receptor proteinpresent in the sample competes with labeled protein for binding to aspecific binding agent, e.g., an antibody specifically reactive with thereceptor protein. The binding agent may be bound to a solid substrate orsurface to effect separation of bound labeled protein from the unboundlabeled protein. Alternately, the competitive binding assay may beconducted in liquid phase and a variety of techniques known in the artmay be used to separate the bound labeled protein from the unboundlabeled protein. Following separation, the amount of bound labeledprotein is determined. The amount of protein present in the sample isinversely proportional to the amount of labeled protein binding.

Alternatively, a homogeneous immunoassay may be performed in which aseparation step is not needed. In these immunoassays, the label on theprotein is altered by the binding of the protein to its specific bindingagent. This alteration in the labeled protein results in a decrease orincrease in the signal emitted by label, so that measurement of thelabel at the end of the immunoassay allows for detection or quantitationof the protein.

Diagnostic detection of receptor proteins may also be performed by avariety of noncompetitive immunoassay methods. For example, a two-site,solid phase sandwich immunoassay may be used. In this type of assay, abinding agent for the protein, e.g., an antibody, is attached to a solidsupport. A second protein binding agent, which may also be an antibody,and which binds the protein at a different site, is labeled. Afterbinding at both sites on the protein has occurred, the unbound labeledbinding agent is removed and the amount of labeled binding agent boundto the solid phase is measured. The amount of labeled binding agentbound is directly proportional to the amount of protein in the sample.

Western blot analysis can be used to determine the presence of receptorproteins in a sample. Electrophoresis is carried out, e.g., on a tissuesample suspected of containing the protein. Following electrophoresis toseparate the proteins, and transfer of the proteins to a suitable solidsupport, e.g., a nitrocellulose filter, the solid support is incubatedwith an antibody reactive with the protein. This antibody may belabeled, or alternatively may be detected by subsequent incubation witha second labeled antibody that binds the primary antibody.

The immunoassay formats described above may employ labeled assaycomponents. The label may be coupled directly or indirectly to thedesired component of the assay according to methods well known in theart. A wide variety of labels and methods may be used. Traditionally, aradioactive label incorporating ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P was used.Non-radioactive labels include ligands which bind to labeled antibodies,fluorophores, chemiluminescent agents, enzymes, and antibodies which canserve as specific binding pair members for a labeled ligand. The choiceof label depends on sensitivity required, ease of conjugation with thecompound, stability requirements, and available instrumentation. For areview of various labeling or signal producing systems which may beused, see U.S. Pat. No. 4,391,904.

Antibodies reactive with a particular protein can also be measured by avariety of immunoassay methods. Thus modifications of the aboveprocedures may be used to determine the amounts or affinities of variousligand analogs or ligand or receptor antibody preparations. For a reviewof immunological and immunoassay procedures applicable to themeasurement of antibodies by immunoassay techniques, see, e.g., Stitesand Terr (eds.) Basic and Clinical Immunology (7th ed.) supra; Maggio(ed.) Enzyme Immunoassay, supra; and Harlow and Lane Antibodies, ALaboratory Manual, supra.

Screens to evaluate the binding and activity of mAbs and bindingcompositions encompass a variety of methods. Binding can be assayed bydetectably labeling the antibody or binding composition as describedabove. Cells responsive to ligand can be used to assay antibody orbinding composition.

To evaluate ligand chemoattraction or chemokinetic ability, experimentalanimals, e.g., mice, are preferably used. Cell counts are made prior toand at various time points after administration of a bolus of thecandidate agonist or antagonist. Levels are analyzed in various samples,e.g., blood, serum, nasal or pulmonary lavages, or tissue biopsystaining. A successful depleting mAb or binding composition will, e.g.,significantly lower the level of chemoattraction of receptor bearingcells. Such may be at least about 10%, preferably at least about 20%,30%, 50%, 70%, or more.

Evaluation of antibodies can be performed in other animals, e.g., humansusing various methods. For example, blood samples are withdrawn frompatients suffering from a potential metastatic disease or disorderbefore and after treatment with a candidate mAb.

IV. Uses

The tissue-selective homing of metastatic tumors has long beenrecognized. Recent advances in the field support a model in which cellhoming is achieved by sequential engagement of differentially expressedand independently regulated vascular and leukocyte adhesion molecules,and signaling receptors and their ligands. Butcher and Picker (1996)Science 272:60-66. The observation that chemokines, a superfamily ofsmall secreted proteins with G protein-coupled receptors (Baggiolini(1998) Nature 392:565-568) can attract leukocytes led to the hypothesisthat chemokines provide key signals directing recruitment of Tlymphocyte subsets into lymphoid and extra-lymphoid immune effectorsites. Analogously, tumor metastasis appears to make use of many similarprocesses, which may be similarly blocked.

A statistically significant change in the numbers of primary tumor ormetastasizing cells will typically be at least about 10%, preferably20%, 30%, 50%, 70%, 90%, or more. The effects may be specific inblocking tumor growth or progression or chemoattraction to specificpoints, or may be chemokinetic, in reducing general movement of cells,but not necessarily in a specific direction, e.g., of concentrationgradient.

The present invention will be use-ful in the treatment of medicalconditions or diseases associated with cancers. See, e.g., Bertino, etal. (eds. 1996) Encyclopedia of Cancer Academic Press; Devita, et al.(eds. 1997) Cancer: Principles & Practice of Oncology Lippincott,Williams and Wilkins; Devita (1997) Principles and Practice of OncologyLippincott Williams and Wilkins; Cavalli, et al. (1996) Textbook ofMedical Oncology Dunitz Martin Ltd; Horwich (ed. 1995) Oncology: AMultidisciplinary Textbook Lippincott-Raven; Peckham, et al. (eds. 1995)Oxford Textbook of Oncology Oxford Univ. Press; Mendelsohn, et al.(1995) The Molecular Basis of Cancer Saunders, Philadelphia; and McArdle(1990) Surgical Oncology: Current Concepts and PracticeButterworth-Heinemann. The specific reagents and antagonists describedmay be combined with other treatments of the medical conditionsdescribed herein, e.g., a chemotherapy, radiation therapy,immunotherapy, or surgical method, including alkylating agents,antimetabolites, antihormones, therapeutic for various symptoms, e.g.,painkillers, diuretics, antidiuretics, antivirals, antibiotics,nutritional supplements, anemia therapeutics, blood clottingtherapeutics, bone therapeutics, and psychiatric and psychologicaltherapeutics.

To prepare pharmaceutical or sterile compositions including, e.g., thedesired antagonist, the material is admixed with a pharmaceuticallyacceptable carrier or excipient which is preferably inert. Preparationof such pharmaceutical compositions is known in the art, see, e.g.,Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: NationalFormulary, Mack Publishing Company, Easton, Pa. (1984). Typically,therapeutic compositions are sterile.

Specific labeling reagents or antagonists, e.g., ligand muteins,antibodies, or binding compositions, are normally administeredparenterally, preferably intravenously. Since such protein or peptideantagonists may be immunogenic they are preferably administered slowly,either by a conventional IV administration set or from a subcutaneousdepot, e.g. as taught by Tomasi, et al., U.S. Pat. No. 4,732,863. Smallmolecule drugs may be orally active, or administered in other standardmethods.

When administered parenterally the therapeutics will typically beformulated in a unit dosage injectable form (solution, suspension,emulsion) in association with a pharmaceutically acceptable parenteralvehicle. Such vehicles are inherently non-toxic and non-therapeutic. Theantagonist may be administered in aqueous vehicles such as water,saline, or buffered vehicles with or without various additives and/ordiluting agents. Alternatively, a suspension, such as a zinc suspension,can be prepared to include the peptide. Such a suspension can be usefulfor subcutaneous (SQ), intradermal (ID), or intramuscular (IM)injection. The proportion of therapeutic entity and additive can bevaried over a broad range so long as both are present in effectiveamounts. The therapeutic is preferably formulated in purified formsubstantially free of aggregates, other proteins, endotoxins, and thelike, at concentrations of about 5 to 30 mg/ml, preferably 10 to 20mg/ml. Preferably, the endotoxin levels are less than 2.5 EU/ml. See,e.g., Avis, et al. (eds. 1993) Pharmaceutical Dosage Forms: ParenteralMedications 2d ed., Dekker, NY; Lieberman, et al. (eds. 1990)Pharmaceutical Dosage Forms: Tablets 2d ed., Dekker, NY; Lieberman, etal. (eds. 1990) Pharmaceutical Dosage Forms: Disperse Systems Dekker,NY; Fodor, et al. (1991) Science 251:767-773; Coligan (ed.) CurrentProtocols in Immunology; Hood, et al. Immunology Benjamin/Cummings; Paul(ed. 1997) Fundamental Immunology 4th ed., Academic Press; Parce, et al.(1989) Science 246:243-247; Owicki, et al. (1990) Proc. Nat'l Acad. Sci.USA 87:4007-4011; and Blundell and Johnson (1976) ProteinCrystallography, Academic Press, New York.

Selecting an administration regimen for a therapeutic agonist orantagonist depends on several factors, including the serum or tissueturnover rate of the therapeutic, the immunogenicity of the therapeutic,the accessibility of the target cells, and the general tolerance of thepatient to the stress of therapy. Preferably, an administration regimenmaximizes the amount of therapeutic delivered to the patient consistentwith an acceptable level of side effects. Accordingly, the amount oftherapeutic delivered depends in part on the particular antagonist andthe severity of the condition being treated. Guidance in selectingappropriate doses of antibodies is found in the literature ontherapeutic uses, e.g. Bach et al., chapter 22, in Ferrone, et al. (eds.1985) Handbook of Monoclonal Antibodies Noges Publications, Park Ridge,N.J.; and Russell, pgs. 303-357, and Smith et al., pgs. 365-389, inHaber, et al. (eds. 1977) Antibodies in Human Diagnosis and TherapyRaven Press, New York, N.Y.

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known in the art to affect treatment orpredicted to affect treatment. Generally, the dose begins with an amountsomewhat less than the optimum dose and it is increased by smallincrements thereafter until the desired or optimum effect is achievedrelative to any negative side effects. Numbers of receptor bearing cellsin defined samples might be important indicators of when an effectivedose is reached. Preferably, an antibody or binding composition thereofthat will be used is derived from the same species as the animaltargeted for treatment, thereby minimizing a humoral response to thereagent.

The total weekly dose ranges for antibodies or fragments thereof, whichspecifically bind to ligand, range generally from about 1 ng, moregenerally from about 10 ng, typically from about 100 ng; more typicallyfrom about 1 μg, more typically from about 10 μg, preferably from about100 μg, and more preferably from about 1 mg per kilogram body weight.Although higher amounts may be more efficacious, the lower dosestypically will have fewer adverse effects. Generally the range will beless than 100 mg, preferably less than about 50 mg, and more preferablyless than about 25 mg per kilogram body weight.

The weekly dose ranges for antagonists, e.g., antibody, bindingfragments, range from about 10 μg, preferably at least about 50 μg, andmore preferably at least about 100 μg per kilogram of body weight.Generally, the range will be less than about 1000 μg, preferably lessthan about 500 μg, and more preferably less than about 100 μg perkilogram of body weight. Dosages are on a schedule which effects thedesired treatment and can be periodic over shorter or longer term. Ingeneral, ranges will be from at least about 10 μg to about 50 mg,preferably about 100 μg to about 10 mg per kilogram body weight.

Other antagonists of the ligands, e.g., muteins, are also contemplated.Hourly dose ranges for muteins range from at least about 10 μg,generally at least about 50 μg, typically at least about 100 μg, andpreferably at least 500 μg per hour. Generally the dosage will be lessthan about 100 mg, typically less than about 30 mg, preferably less thanabout 10 mg, and more preferably less than about 6 mg per hour. Generalranges will be from at least about 1 μg to about 1000 μg, preferablyabout 10 μg to about 500 μg per hour.

The phrase “effective amount” means an amount sufficient to effect adesired response, or to ameliorate a symptom or sign, e.g., ofmetastasis or primary tumor progression, size, or growth. Typicalmammalian hosts will include mice, rats, cats, dogs, and primates,including humans. An effective amount for a particular patient may varydepending on factors such as the condition being treated, the overallhealth of the patient, the method, route, and dose of administration andthe severity of side affects. Preferably, the effect will result in achange in quantitation of at least about 10%, preferably at least 20%,30%, 50%, 70%, or even 90% or more. When in combination, an effectiveamount is in ratio to a combination of components and the effect is notlimited to individual components alone.

An effective amount of therapeutic will modulate the symptoms typicallyby at least about 10%; usually by at least about 20%; preferably atleast about 30%; or more preferably at least about 50%. Alternatively,modulation of migration will mean that the migration or trafficking ofvarious cell types is affected. Such will result in, e.g., statisticallysignificant and quantifiable changes in the numbers of cells beingaffected. This may be a decrease in the numbers of target cells beingattracted within a time period or target area. Rate of primary tumorprogression, size, or growth may also be monitored.

The present invention provides reagents which will find use intherapeutic applications as described elsewhere herein, e.g., in thegeneral description for treating neoplastic disorders. See, e.g., Berkow(ed.) The Merck Manual of Diagnosis and Therapy, Merck & Co., Rahway,N.J.; Thorn, et al. Harrison's Principles of Internal Medicine,McGraw-Hill, NY; Gilman, et al. (eds. 1990) Goodman and Gilman's: ThePharmacological Bases of Therapeutics, 8th Ed., Pergamon Press;Remington's Pharmaceutical Sciences, 17th ed. (1990), Mack PublishingCo., Easton, Pa.; Langer (1990) Science 249:1527-1533; and Merck Index,Merck & Co., Rahway, N.J.

Moreover, antisense nucleic acids may be used. For example, antisensepolynucleotides against the ligand encoding nucleic acids may functionin a manner like ligand antagonists, and antisense against the receptormay function like receptor antagonists. Thus, it may be possible toblock the signaling through the pathway with antisense nucleic acids.Conversely, nucleic acids for the receptor may serve as agonists,increasing the numbers of receptor on the cell, thereby increasing cellsensitivity to ligand.

Other methods based upon these observations may be developed. Theattraction may be effected to specific sites where treatment may be moreeffective. Gradients may be set up to attract the metastatic cells tosites of effective treatment, or to trap metastatic cells for easyremoval. Conversely, receptor desensitization might be effected byflooding the system with huge excesses of the cognate ligands in definedtemporal patterns. Receptor targeting may allow for specificadministration of therapeutic drugs, e.g., by localized attraction,activation, absorption, or activation of killing, etc.

The broad scope of this invention is best understood with reference tothe following examples, which are not intended to limit the inventionsto the specific embodiments.

Examples I. General Methods

Some of the standard methods are described or referenced, e.g., inManiatis, et al. (1982) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual, (2d ed.), vols. 1-3, CSHPress, NY; Ausubel, et al., Biology, Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements) CurrentProtocols in Molecular Biology, Greene/Wiley, New York; Innis, et al.(eds.)(1990) PCR Protocols: A Guide to Methods and Applications AcademicPress, N.Y. Methods for protein purification include such methods asammonium sulfate precipitation, column chromatography, electrophoresis,centrifugation, crystallization, and others. See, e.g., Ausubel, et al.(1987 and periodic supplements); Deutscher (1990) “Guide to ProteinPurification” in Methods in Enzymology, vol. 182, and other volumes inthis series; manufacturer's literature on use of protein purificationproducts, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond,Calif.; and Coligan, et al. (eds.) (1995 and periodic supplements)Current Protocols in Protein Science, John Wiley & Sons, New York, N.Y.Combination with recombinant techniques allow fusion to appropriatesegments, e.g., to a FLAG sequence or an equivalent which can be fusedvia a protease-removable sequence. See, e.g., Hochuli (1989) ChemischeIndustrie 12:69-70; Hochuli (1990) “Purification of Recombinant Proteinswith Metal Chelate Absorbent” in Setlow (ed.) Genetic Engineering,Principle and Methods 12:87-98, Plenum Press, N.Y.; and Crowe, et al.(1992) QIAexpress: The High Level Expression & Protein PurificationSystem QIAGEN, Inc., Chatsworth, Calif.

Standard immunological techniques are described, e.g., in Hertzenberg,et al. (eds. 1996) Weir's Handbook of Experimental Immunology vols. 1-4,Blackwell Science; Coligan (1991) Current Protocols in ImmunologyWiley/Greene, NY; and Methods in Enzymology volumes. 70, 73, 74, 84, 92,93, 108, 116, 121, 132, 150, 162, and 163.

Cell migration assays are performed as previously described, e.g., inBacon, et al. (1988) Br. J. Pharmacol. 95:966-974. Other traffickingassays are also available. See, e.g., Quidling-Järbrink, et al. (1995)Eur. J. Immunol. 25:322-327; Koch, et al. (1994) J. ClinicalInvestigation 93:921-928; and Antony, et al. (1993) J. Immunol.151:7216-7223.

Alternatively, an activation assay or attraction assay is used. Anappropriate cell type is selected, e.g., hematopoietic cells, myeloid(macrophages, neutrophils, polymorphonuclear cells, etc.) or lymphoid (Tcell, B cell, or NK cells), neural cells (neurons, neuroglia,oligodendrocytes, astrocytes, etc.), or stem cells, e.g., progenitorcells which differentiate to other cell types, e.g., gut crypt cells andundifferentiated cell types.

Chemokines may also be assayed for activity in hemopoietic assays asdescribed, e.g., by H. Broxmeyer. See Bellido, et al. (1995) J. ClinicalInvestigation 95:2886-2895; and Jilka, et al. (1995) Expt'l Hematology23:500-506. They may be assayed for angiogenic activities as described,e.g., by Streiter, et al. (1992) Am. J. Pathol. 141:1279-1284. Or for arole in inflammation. See, e.g., Wakefield, et al. (1996) J. SurgicalRes. 64:26-31.

Other assays will include those which have been demonstrated with otherchemokines. See, e.g., Schall and Bacon (1994) Current Opinion inImmunology 6:865-873; and Bacon and Schall (1996) Int. Arch. Allergy &Immunol. 109:97-109. Ca2+ flux upon chemokine stimulation is measuredaccording to the published procedure described in Bacon, et al. (1995)J. Immunol. 154:3654-3666.

FACS analyses are described in Melamed, et al. (1990) Flow Cytometry andSorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988) Practical FlowCytometry Liss, New York, N.Y.; and Robinson, et al. (1993) Handbook ofFlow Cytometry Methods Wiley-Liss, New York, N.Y.

II. Cell Culture and Tissue Samples

Human primary cells were obtained, e.g., from the ATCC and clinicalcollaborators. Additional cell cultures and tumor samples are collected.Clinical sources are available, e.g., Cooperative Human Tissue Network,NIH.

Panels of breast cancer, head and neck, and melanoma cell lines werecollected. Additional target primary cell lines to be collected include,e.g., prostate cancer, gastrointestinal cancer, and lung cancer celllines.

Tissue samples are also collected from primary tumors. Primate, e.g.,human, are preferred, but in many circumstances, mouse or other speciesmay be collected. Metastatic tumors will also be evaluated, both forreceptor expression, and for chemokine chemoattraction assays.Additional tumors to be evaluated include, e.g., prostate,gastrointestinal, and lung cancers.

Also, chemokine expression will be evaluated for lymph node, bonemarrow, brain, liver, skin, and lung. Those chemokines which areexpressed therefrom are candidate chemoattractants for cancers whichmetastasize to those organs. Antagonists of the ligand and receptorshould be tested for blockage of metastasis to those organs.

III. Isolation of Encoding Sequences

Human, mouse, or rat chemokine receptor or chemokine sequences arereadily available. See, e.g., GenBank and Derwent patent sequencedatabases. Appropriate PCR primers or hybridization probes can beselected. Sequences will be useful for evaluation of expression ofreceptors in tumor cells or chemokines in target organs. Isolated ligandsequences will serve as starting points for mutagenesis efforts toidentify mutein antagonists. Gene sequences may be useful to producerecombinant protein for antibody production.

IV. Distribution Analysis

For Southern blotting, 5 μg of each cDNA library is digested with theappropriate restriction enzymes to release the insert, subjected to gelelectrophoresis, and transferred to Hybond-N⁺ membrane. For Northernblotting, RNAs are isolated using RNAzol B (TEL-TEST, Inc.) and analyzedby electrophoresis on a 1% formaldehyde-agarose gel and transferred toHybond-N⁺ membrane. Northern and Southern blots are hybridized, e.g.,for 16 hr at 65° C. with ³²P-labeled probes obtained by randomly priming(Prime-it; Stratagene) the full length inserts. After hybridization,blots are washed at high stringency and exposed to film.

PCR methods have been applied, and chemokine or receptor specificprimers may be designed. Diagnostic methods are well known. Quantitativetechniques are also available, e.g., TAQMAN™. Putative metastatic tumorcells will be evaluated for expression of chemokine receptors, which maymediate metastatic attraction to specific target organs. Conversely,target organs to which metastasis is common may express chemokines whichserve to chemoattract metastatic cells to them. Thus, evaluation of thechemokine production patterns by target organs may explain the tumorswhich are chemoattracted to establish in the secondary site.

Diagnosis of receptor expression by a primary tumor may provide guidanceas to what tumors may be susceptible to blockage of metastasis by whichantagonists. This may be useful in determining therapeutic treatmentstrategies, e.g., what tumors may be effectively treated for metastaticblockage. Moreover, specific expression of particular receptors mayserve as markers for targeting therapeutic reagents to the differenttumor types.

Nucleic acid expression analysis, e.g., PCR, Taqman, hybridization data,and/or RNA protection, is preferably confirmed by evaluating proteinexpression. This may be in the form of protein assay, e.g., Westernprotein blotting techniques, immunoassay, or immunohistochemistry.Statistical analysis will be useful in determining the likelihood ofefficacy of antagonist treatment.

Additional confirmatory methods include, e.g., migration and invasionassays, F-actin polymerization assays, and cell motility evaluation.Transfection models may be applied to confer trafficking of otherwiseinert cell types. Studies may be based upon rodent models, etc.

V. Chemoattraction Assays

Recombinant chemokine is produced, e.g., in E. coli and purified. SeeHedrick, et al. (1998) Blood 91:4242-4247. A modified Boyden chamber isused. Human tumor cells, e.g., lines or primary or secondary tumors, inDMEM, pH 6.9, 0.1% bovine serum albumin, are added to the top chamber of8 μm pore polycarbonate Transwell culture insert (Costar) and incubatedwith the indicated concentrations of purified chemokine in the bottomchamber for 6-24 h. The number of migrating cells of each subtype isdetermined, e.g., by staining and counting. See, e.g., Youngs, et al.(1997) Int. J. Cancer 71:257-266.

Chemotaxis assays are performed with, e.g., human tumor cells. Celllines or primary tumor cells will be evaluated from breast, head andneck, melanoma, prostate, gastrointestinal, and lung cancers. Other celltypes express the various chemokine receptors. Recombinant chemokineshould have effects on the cell types expressing receptor.

VI. Antibody Production

Appropriate mammals are immunized with appropriate amounts, e.g., ofchemokine gene transfected cells, e.g., intraperitoneally every 2 weeksfor 8 weeks. Similar methods may be used to produce antibodies whichbind to receptor, e.g., CCR7, CXCR4, or GPR2, polypeptides, ortransfected cells expressing the receptor may be used. Typically,rodents are used, though other species should accommodate production ofselective and specific antibodies. The final immunization is givenintravenously (IV) through the tail vein.

Generic polyclonal antibodies may be collected. Alternatively,monoclonal antibodies can be produced. For example, four days after theIV injection, the spleen is removed and fused to SP2/0 and NS1 cells.HAT resistant hybridomas are selected, e.g., using a protocol designedby Stem Cell Technologies (Vancouver, BC). After 10 days of HATselection, resistant foci are transferred to 96 well plates and expandedfor 3 days. Antibody containing supernatants are analyzed, e.g., by FACSfor binding to NIH3T3/surface MIP-3β transfectants. Many differentMIP-3β mAbs are typically produced. Those antibodies may be isolated andmodified, e.g., by labeling or other means as is standard in the art.See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual CSHPress; Goding (1986) Monoclonal Antibodies: Principles and Practice (2ded.) Academic Press, New York, N.Y. Methods to conjugate magneticreagents, toxic entities, labels, attach the antibodies to solidsubstrates, to sterile filter, etc., are known in the art.

VII. Purification of Cells

Chemokine responsive cells may be identified using the reagentsdescribed herein. For example, cells which are chemoattracted towards,e.g., SDF-1 may be purified from other cells by collecting those cellswhich traverse towards SDF-1. Such chemotaxis may be to a source ofchemokine, or may be across a porous membrane or other substrate. Seeabove, in the microchemotaxis assay.

Alternatively, responsive cells may be identified by expression of thereceptor, e.g., CXCR4, as provided herein. Thus, antibodies whichrecognize CXCR4 may be used as a positive marker for sorting cellslikely to respond to SDF-1, and thus be chemoattracted to bone marrow.Conversely, the marker may be used to deplete CXCR4 bearing cells, e.g.,by magnetic depletion or toxic conjugates.

Analysis of human samples can be evaluated in a similar manner. Abiological sample, e.g., blood, tissue biopsy sample, lung or nasallavage, skin punch, is obtained from an individual suffering from aneoplastic related disorder. Chemokine responsive cell analysis isperformed, e.g., by FACS analysis, or similar means. And those cells maybe primary tumor cells which are to be targeted, e.g., by toxicconjugates.

VIII. Chemokine Antagonists

Various antagonists of designated chemokines are made available. Forexample, antibodies against the chemokine itself may block the bindingof ligand to its receptor, thereby serving as a direct receptorantagonist. Other antagonists may function by blocking the binding ofligand to receptor, e.g., by binding to the receptor in a way topreclude the possibility of binding of ligand. Other antagonists, e.g.,mutein antagonists or aptamers, may bind to the receptor withoutsignaling, thereby blocking a true agonist from binding. Many of thesemay serve to block the signal transmitted to target cells, e.g.,specifically SDF-1-responsive cells. Small molecule compounds may alsobe identified which block interaction of ligand with receptor.

Information on the criticality of particular residues is determined,e.g., using standard procedures and analysis. Standard mutagenesisanalysis is performed, e.g., by generating many different variants atdetermined positions, e.g., at the positions likely involved in receptorbinding, and evaluating biological activities of the variants. This maybe performed to the extent of determining positions which modifyactivity, or to focus on specific positions to determine the residueswhich can be substituted to either retain, block, or modulate biologicalactivity.

Alternatively, analysis of natural variants can indicate what positionstolerate natural mutations. This may result from populational analysisof variation among individuals, or across strains or species. Samplesfrom selected individuals are analyzed, e.g., by PCR analysis andsequencing. This allows evaluation of population polymorphisms.

IX. Labeling of Cells

With specific antibodies, not necessarily functionally blocking,labeling of cells may be performed. Antibodies of appropriatespecificity can, e.g., be labeled with a detectable signal. Fluorescentantibodies are a common example.

Certain cells may be labeled in solution, e.g., individual cellsexpressing surface antigens. FACs sorting is based mostly on thisproperty. Other cells may be labeled in tissue form, e.g.,immunohistochemistry. The means to get antibody into fixed tissues areknown, and similar methods may be used in unfixed tissues, ex vivo, orin vivo. See, e.g., Young and Heath (eds. 2000) Wheater's FunctionalHistology: A Text and Colour Atlas (Book with CD-ROM) ChurchillLivingstone; Kerr (1999) Atlas of Functional Histology Mosby; and Ross,et al. (eds. 1995) Histology: A Text and Atlas Lippincott, Williams &Wilkins.

In situ cellular labeling of primary or secondary tumors by antibody maybe used to induce natural killing mechanisms, e.g., antigen dependentcell-mediated cytotoxicity (ADCC), complement mediated cell lyticprocesses, or opsinization and macrophage phagocytosis, among others.Specific localization of the receptor markers with antibodies may beuseful in both diagnostic and therapeutic contexts. Diagnostically, theantibodies may allow localization of receptor expressing primary tumorcells, e.g., by use of radio-opaque labels, to determine the locationand extent of tumor growth or metastasis. The antibodies may be used tolocalize an activating agent, e.g., as alkaline phosphatase is localizedand acts on substrate, similar to that mechanism used forimmunohistochemistry applications. A similar strategy may be used tolocally enzymatically activate a killing process, e.g., to activate anotherwise inert toxin. For example, an inactive lectin (pro-lectin) maybecome proteolytically activated by an antibody enzyme conjugate. Or, anenergy absorbing reagent may be conjugated to the antibody, therebyspecifically localizing the reagent which absorbs energy to cause tissuein proximity to become subject to concentration of an external radiationsource.

All citations herein are incorporated herein by reference to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited bythe terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled; and the invention is notto be limited by the specific embodiments that have been presentedherein by way of example.

1. A method of inhibiting metastasis of a cell, said method comprisingblocking signaling of a chemokine receptor on said cell.
 2. The methodof claim 1, wherein said metastasis is organ specific.
 3. The method ofclaim 1, wherein said metastasis is to the lymph node, bone marrow, orskin.
 4. The method of claim 3, wherein said metastasis is to the lymphnode.
 5. The method of claim 1, wherein said cell is a carcinoma cell,including a breast, head and neck, melanoma, or prostate.
 6. The methodof claim 1, wherein said carcinoma cell is breast or melanoma.
 7. Themethod of claim 1, wherein said chemokine receptor is CCR7, CXCR4, orCCR10.
 8. The method of claim 7, wherein said blocking signaling is withan antibody against said chemokine receptor.
 9. The method of claim 7,wherein said chemokine receptor is CCR7, and said blocking is with: a)an antibody against FSEC, CKP9, or CKpll; or b) an antagonist mutein ofFSEC, CKP9, or CKpll.
 10. The method of claim 7, wherein said chemokinereceptor is CXCR4, and said blocking is with: a) an antibody againstSDF-1; or b) an antagonist mutein of SDF-1.
 11. The method of claim 7,wherein said chemokine receptor is GPR2, and said blocking is with: a)an antibody against CTACK or Vic; or b) an antagonist mutein of CTACK orVic.
 12. The method of claim 1, wherein said blocking signaling is with:a) an antibody against said chemokine receptor; or b) a drug whichinhibits signaling of said chemokine receptor including pertussis toxin.13. The method of claim 1, which is combined with another treatment forcancer.
 14. The method of claim 13, wherein said treatment is selectedfrom chemotherapy, radiation therapy, immunotherapy, and surgery. 15.The method of claim 13, wherein said treatment is before said blocking.16. A method of screening for chemokine receptors on primary tumor ormetastatic cells, comprising identifying which chemokine receptors areexpressed on said cell.
 17. The method of claim 16, wherein saididentifying: a) is antibody labeling, ligand testing, or PCR analysis;or b) results in determination of therapeutic treatment.
 18. Acomposition comprising an antitumor agent and: a) a chemokine receptorantagonist; or b) a toxic conjugate targeted to a chemokine receptor.19. The composition of claim 18, wherein said antagonist or toxicconjugate is selected from: a) an antibody against FSEC, CKβ9, or CKβ11;b) an antagonist mutein of FSEC, CKβ9, or CKβ11; c) an antibody againstSDF-1; d) an antagonist mutein of SDF-1; e) an antibody against CTACK orVic; f) an antagonist mutein of CTACK or Vic; g) an antibody againstsaid chemokine receptor; or h) a drug which inhibits signaling of saidchemokine receptor including pertussis toxin.
 20. A method of treatingcancer in an animal, comprising administering to said animal aneffective amount of the composition of claim 18.